Preparation method and application of fructus cannabis protein peptide with sugar absorption blocking effect

Abstract

The invention provides a preparation method and application of fructus cannabis protein peptide with a sugar absorption blocking effect, and belongs to the technical field of functional active peptides, and the preparation method comprises the following steps: crushing fructus cannabis, and degreasing through low-temperature continuous phase change to obtain degreased fructus cannabis meal after degreasing; the degreased hemp seed meal is hydrolyzed through polysaccharase and protease in sequence, low-temperature salting-out is carried out after hydrolysis is finished, drying and sterilization are carried out, and the hemp seed protein peptide is obtained. The fructus cannabis protein peptide obtained by the technical scheme and the preparation method disclosed by the invention has the following effects: 1, the fructus cannabis protein peptide prepared by the preparation method disclosed by the invention can be used for remarkably inhibiting the activity of alpha-amylase and alpha-glycosidase (the maximum inhibition rates can respectively reach 84.2% and 75.1%), so that digestion and absorption of a human body on carbohydrates such as starch, dextrin and disaccharide are blocked; and 2, double-blind clinical test results show that the fructus cannabis protein peptide prepared by the invention can effectively reduce the postprandial blood sugar peak value and peak area, and the effective sugar breaking rate can reach 30-60%.

Description

Technical Field

[0001] The present invention relates to the technical field of functional active peptide products, and particularly relates to a preparation method and application of hemp seed protein peptide with a function of blocking sugar absorption. Background Art

[0002] With the high incidence of chronic diseases such as diabetes and obesity, the importance of blood sugar control has become increasingly prominent, and more and more people have begun to try to control blood sugar by cutting off sugar. Generally speaking, cutting off sugar refers to strictly restricting the intake of carbohydrates, or blocking the digestion and absorption of sugar substances through amylase and glycosidase inhibitors to achieve the purpose of blood sugar control. In addition, cutting off sugar also helps to improve indicators such as blood lipids, thereby reducing the incidence of cardiovascular diseases; in terms of body management, blocking sugar absorption can reduce calorie intake and prompt the body to consume fat to provide energy, so it is also favored by many weight loss people. Therefore, in today's society, cutting off sugar has gradually become a much - concerned dietary trend.

[0003] With the continuous improvement of people's demand for healthy diets, the food field is constantly seeking healthier and more effective natural active ingredients. Hemp seed protein peptide is a small - molecule active peptide prepared from hemp seed protein through a series of enzymatic hydrolysis processes, and it has been confirmed by many studies to have various activities such as antioxidant, blood - pressure - lowering, immunity - enhancing, and anti - fatigue effects.

[0004] Chinese Patent with publication number CN112410393B and title "A Hemp Active Peptide and Its Preparation Method and Application" discloses that first, pre - cooled defatted hemp seed meal and dry ice are mixed and pulverized to obtain hemp seed meal powder, then water and calcium carbonate are added to prepare an alkaline suspension, followed by grinding, sieving, then two - step enzymatic hydrolysis using a mixed protease and a neutral protease in sequence, and finally inactivating the enzyme, filtering, centrifuging, decolorizing, and sterilizing to obtain the hemp active peptide. The hemp active peptide prepared by the above - mentioned scheme has high purity, full taste, and high antioxidant activity, and has the effects of antioxidant, blood - pressure - lowering, anti - fatigue, and improving exercise tolerance. However, the products in the above - mentioned technical scheme and the existing technology only focus on functions such as antioxidant and blood - pressure - lowering. Summary of the Invention

[0005] In view of this, the present invention provides a preparation method and application of hemp seed protein peptide with a function of blocking sugar absorption. The hemp seed protein peptide obtained by the technical scheme and preparation method of the present invention can significantly inhibit the activities of α - amylase and α - glycosidase, thereby blocking the digestion and absorption of starch and sucrose by the human body. Clinical test results show that the hemp seed protein peptide prepared by the present invention can effectively reduce the post - meal blood sugar peak value and peak area, and the effective sugar - cutting rate can reach 30 - 60%, providing a new direction for the development of hemp seeds.

[0006] To achieve the above object, the present invention provides a method for preparing hemp seed protein peptide with the function of blocking sugar absorption, comprising the following steps: crushing hemp seeds, degreasing them by low-temperature continuous phase transition, obtaining defatted hemp seed meal after degreasing, hydrolyzing the defatted hemp seed meal successively with polysaccharide enzyme and protease, performing low-temperature salting out after hydrolysis, and obtaining hemp seed protein peptide after drying and sterilization.

[0007] Optionally, the extractant for low-temperature continuous phase transition is No. 15 solvent oil; the mass-volume ratio of hemp seeds to the extractant is 1:8 - 15; the low-temperature continuous phase transition includes the following conditions: the temperature is 20°C - 35°C, the pressure is 2.5 Mpa - 4 Mpa, the flow rate is 50 - 80 L / h, the continuous extraction time is 90 min - 120 min, the desorption temperature is 70°C - 85°C, and the desorption pressure is 0.5 Mpa - 1 Mpa.

[0008] Optionally, the polysaccharide enzyme hydrolysis includes the following steps: mixing defatted hemp seed meal with water, the solid-liquid ratio is 1:10 - 30, the mass of the added polysaccharide enzyme is 10 - 15% of the substrate mass, adjusting the pH to 3.0 - 5.5, and the hydrolysis duration is 1 - 4 h; the substrate is defatted hemp seed meal and water.

[0009] Optionally, the polysaccharide enzyme is one or a combination of two or more of cellulase, pectinase, amylase, glycosidase, hemicellulose, glucanase, and xylanase.

[0010] Optionally, the protease hydrolysis includes the following steps: inactivating the enzyme of the product after polysaccharide enzyme hydrolysis, then adding protease, the addition amount of protease is 2 - 6% of the mass of the product after polysaccharide enzyme hydrolysis, adjusting the pH to 6.0 - 8.5 for hydrolysis, and the hydrolysis duration is 15 - 35 min; the protease is bacillus licheniformis protease.

[0011] Optionally, the low-temperature salting out is to centrifuge and filter the product after protease hydrolysis to obtain a filtrate, cool it to 2 - 4°C, continuously add saturated ammonium sulfate solution to the filtrate until protein flocculation occurs, perform salting out for 30 - 90 min, and take the precipitate after centrifugation.

[0012] Optionally, the drying is spray drying, including the following steps: performing spray drying using a spray dryer, the inlet air temperature is 15 - 25°C, the outlet air temperature is 30 - 40°C, the feed rate is 50 - 150 kg / h, the voltage of the electrostatic generator is 30 - 55 kV, and the pressure of the atomizer is 15 - 20 MPa.

[0013] Optionally, the sterilization is ultra-high pressure sterilization, including the following steps: when performing ultra-high pressure sterilization, the working pressure is 100 - 500 MPa, the working temperature is 25 - 40°C, and the pressure holding time is 20 - 90 s.

[0014] To achieve the above object, the present invention also provides a hemp seed protein peptide having a sugar-breaking effect, which is prepared by a preparation method of hemp seed protein peptide and comprises the following short peptide sequences: SEQ ID NO: 1: Met-Thr-Met-Trp, SEQ ID NO: 2: Tyr-Lys-Pro-Val-Tyr, SEQ ID NO: 3: Thr-Pro-Val-Ser-Ile-Leu, SEQ ID NO: 4: Met-Ala-His-Leu-Phe, SEQ ID NO: 5: Met-Val-Ser-Leu-Tyr.

[0015] To achieve the above object, the present invention also provides an application of a hemp seed protein peptide prepared by a preparation method of a hemp seed protein peptide having a sugar absorption-blocking effect in a food for blocking sugar absorption.

[0016] The above technical solutions of the present invention at least include the following beneficial effects: 1. The low-temperature continuous phase change deep degreasing technology is used in the technical solution of the present invention: Almost all the raw materials of the existing hemp seed protein peptide products are hemp meal after oil extraction, and unprocessed hemp seeds cannot be directly used because hemp seeds contain 35-45% of oil. If it is directly used for protein extraction or direct enzymatic hydrolysis, it will cause too high oil content in the peptide product, affecting the product taste and storage stability. In addition, the high oil content in the preparation process will also cause problems such as equipment adhesion and blockage, increasing labor and maintenance costs. In the present invention, the low-temperature continuous phase change technology is first applied to the hemp seed degreasing process. After degreasing by this step, the oil content of the hemp meal is reduced to 1-3%, and the oil content of the finished hemp seed protein peptide is reduced to 3-5‰. 2. The step-by-step hydrolysis of polysaccharidase and protease is used in the technical solution of the present invention: Most of the existing production processes of hemp seed protein peptide products use the alkali extraction and acid precipitation method or single enzyme hydrolysis to extract hemp protein for subsequent peptide product production. Due to the use of a strong alkaline environment in alkali extraction and acid precipitation, it is easy to cause changes in the spatial structure of proteins, thereby losing their original biological activity. Enzymatic extraction can maximize the preservation of the natural structure and biological activity of proteins, thus ensuring their medicinal value. However, in some cases, the action of the enzyme may not be complete, resulting in a low extraction rate. Therefore, the present invention first introduces a composite polysaccharide hydrolase and Bacillus licheniformis protease into the production process. The combined action of multiple polysaccharidases can effectively hydrolyze the cellulose, pectin and other difficult-to-hydrolyze polysaccharides in hemp seed cells, break the cell wall to promote protein release; while the protease can efficiently preliminarily degrade some of the macromolecular proteins that have not been completely released into the solution, further improving the protein extraction rate.

[0017] 3. In the technical solution of the present invention, low-temperature salting out is used: Most of the existing preparation processes of hemp seed protein peptides obtain hemp protein through acid precipitation. However, acid precipitation will cause partial denaturation of hemp protein. In addition, the direct discharge of the waste liquid after acid precipitation will cause pollution, and the treatment and discharge will increase the production cost. The present invention uses low-temperature salting out to obtain hemp protein, which not only ensures the natural activity of hemp protein, but also avoids environmental pollution to a certain extent.

[0018] In summary, the hemp seed protein peptide obtained by the combined action of multiple technologies in the technical solution provided by the present invention can significantly inhibit the activities of α-amylase and α-glucosidase, thereby blocking the digestion and absorption of starch and sucrose by the human body. The clinical test results show that the hemp seed protein peptide prepared by the present invention can effectively reduce the postprandial blood glucose peak value and peak area, and the effective sugar-breaking rate can reach 30-60%. Brief Description of the Drawings

[0019] Figure 1 It is the inhibition diagram of hemp seed protein peptide on amylase activity in Example 3 of the present invention; Figure 2 It is the inhibition diagram of hemp seed protein peptide on glucosidase activity in Example 3 of the present invention; Figure 3 It is the schematic diagram of the postprandial blood glucose fluctuation parameters of the volunteers monitored continuously in the verification test of the present invention; Figure 4 It is the schematic diagram of the postprandial blood glucose fluctuation parameters of the volunteers monitored continuously after breakfast in the verification test of the present invention; Figure 5 It is the schematic diagram of the postprandial blood glucose fluctuation parameters of the volunteers monitored continuously after afternoon tea in the verification test of the present invention.

[0020] Figure 1 , 2 , 4, and 5, the hemp peptide and hemp seed peptide are abbreviations of hemp seed protein peptide, representing hemp seed protein peptide. Detailed Embodiments

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art belong to the scope of protection of the present invention.

[0022] Example 1 1000 g of hemp seeds are mechanically shelled and then crushed. Using solvent oil No. 15, with a material-liquid ratio of 1:8, low-temperature continuous phase change degreasing is carried out. The extraction temperature is 20 °C, the extraction pressure is 2.5 Mpa, and it flows through the extraction kettle at a speed of 50 L / h for continuous extraction for 90 min. The desorption temperature is 70 °C, and the desorption pressure is 0.5 Mpa to obtain defatted hemp meal (oil content ≤ 3%).

[0023] The defatted hemp meal is mixed with water at a material-liquid ratio of 1:10, and a compound polysaccharide enzyme (cellulase, pectinase, amylase, glycosidase, ratio 2:2:1:1) accounting for 10‰ of the substrate mass is added. The pH is adjusted to 3.0 for hydrolysis treatment for 1 h. The enzyme inactivation temperature is 85 °C and the time is 5 min. Then, 2.4 L of Bacillus licheniformis high-efficiency protease accounting for 2‰ of the substrate mass is added to the hydrolysis solution, the pH is adjusted to 6.0 for hydrolysis treatment for 15 min. The enzyme inactivation temperature is 85 °C and the time is 5 min. After centrifugal filtration, the solution is cooled to 4 °C, and saturated ammonium sulfate solution is continuously added to the obtained extract until protein flocculation occurs, and salting out is carried out for 30 min. After centrifugal filtration, hemp seed protein peptides are obtained. The hemp seed protein peptides are dried into powder using a spray dryer. The inlet air temperature of the spray drying is 15 °C, the outlet air temperature is 30 °C, the feed rate is 50 kg / h, and the pressure of the atomizer is 15 MPa. Hemp seed protein peptide dry powder is obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0024] The finished product of hemp seed protein peptides is sterilized using an ultra-high pressure sterilization kettle. The working pressure is 100 - 500 MPa, the working temperature is 25 - 40 °C, and the pressure holding time is 20 - 90 s. After sterilization, it is stored at room temperature.

[0025] Example 2 Compared with Example 1, the difference is only that during polysaccharide enzyme hydrolysis, the material-liquid ratio is 1:15, and a compound polysaccharide enzyme (cellulase, pectinase, amylase, glycosidase, ratio 2:2:1:1) accounting for 15‰ of the substrate mass is added. The pH is adjusted to 5.5 for hydrolysis treatment for 4 h. The enzyme inactivation temperature is 85 °C and the time is 5 min. Then, 2.4 L of Bacillus licheniformis high-efficiency protease accounting for 2‰ of the substrate mass is added to the hydrolysis solution, the pH is adjusted to 6.0 for hydrolysis treatment for 15 min. The enzyme inactivation temperature is 85 °C and the time is 5 min. After centrifugal filtration, the solution is cooled to 4 °C, and saturated ammonium sulfate solution is continuously added to the obtained extract until protein flocculation occurs, and salting out is carried out for 90 min. After centrifugal filtration, hemp seed protein peptides are obtained. The remaining steps and operations are the same as those in Example 1.

[0026] Example 3 Compared with Example 1, the only difference is that when the polysaccharide is hydrolyzed, the solid-liquid ratio is 1:20, and a composite polysaccharide enzyme (cellulase, pectinase, amylase, glycosidase, ratio 2:2:1:1) of 10‰ of the substrate mass is added, and the pH is adjusted to 4.5, and the hydrolysis treatment is carried out for 3h. The enzyme inactivation temperature is 85°C and the time is 5min. Continue to add 2‰ of the substrate mass of Bacillus licheniformis protease to the hydrolyzate, adjust the pH to 7.5, and hydrolyze for 15min. The enzyme inactivation temperature is 85°C and the time is 5min. Centrifuge and filter, cool the solution to 4°C, continue to add saturated ammonium sulfate solution to the extract until the protein flocculates, salt out for 30min, and obtain hemp seed protein peptide after centrifugation. The remaining steps and operations are consistent with Example 1.

[0027] Comparative Example 1 1000g of hemp seed was mechanically shelled and then crushed. The hemp meal was defatted using a mechanical oil press and the oil was pressed twice continuously. The defatted hemp meal was mixed with water, with a solid-liquid ratio of 1:20, and a composite polysaccharide enzyme (cellulase, pectinase, amylase, glycosidase, ratio 2:2:1:1) of 10‰ of substrate mass was added, and the pH was adjusted to 4.5 for hydrolysis for 3h. The enzyme inactivation temperature was 85℃ for 5min. 2.4L of Bacillus licheniformis high-efficiency protease of 2‰ of substrate mass was continued to be added to the hydrolyzate, and the pH was adjusted to 7.5 for hydrolysis for 15min. The enzyme inactivation temperature was 85℃ for 5min. Centrifugal filtration was performed, the solution was cooled to 4℃, and a saturated ammonium sulfate solution was continuously added to the extract until the protein flocculated, and salted out for 30min. Hemp seed protein peptide was obtained after centrifugal filtration. The hemp seed protein peptide liquid was dried and powdered using a spray dryer, with the spray drying air inlet temperature being 15°C, the air outlet temperature being 30°C, the feed rate being 50kg / h, and the atomizer pressure being 15MPa. The hemp seed protein peptide dry powder was obtained and vacuum-sealed and packaged using a sterile aluminum foil bag.

[0028] The finished product of hemp seed protein peptide is sterilized in an ultra-high pressure autoclave with a working pressure of 100-500MPa, a working temperature of 25-40°C, a pressure holding time of 20-90s, and is stored at room temperature after sterilization.

[0029] Comparative Example 2 1000g of hemp seeds were mechanically shelled and then crushed. No. 15 solvent oil was used, the solid-liquid ratio was 1:10, low-temperature continuous phase change degreasing was performed, the extraction temperature was 30°C, the extraction pressure was 3.2Mpa, the extraction was carried out at a rate of 50L / h through the extraction kettle, the extraction was continuous for 90min, the decomposition temperature was 70°C, and the decomposition pressure was 0.7Mpa, and defatted hemp meal (oil content ≤3%) was obtained.

[0030] Mix the defatted hemp seed meal with water at a material-liquid ratio of 1:20. Add a compound polysaccharide enzyme (cellulase, pectinase, amylase, glycosidase, with a ratio of 2:2:1:1) at 10‰ of the substrate mass, adjust the pH to 4.5, and conduct hydrolysis treatment for 3 hours. The enzyme inactivation temperature is 85°C and the time is 5 minutes. Centrifuge and filter, cool the solution to 4°C, continuously add saturated ammonium sulfate solution to the obtained extract until protein flocculation occurs, perform salting out for 30 minutes, and obtain hemp seed protein peptides after centrifugation and filtration. Use a spray dryer to dry the hemp seed protein peptide solution into powder. The inlet temperature of the spray dryer is 15°C, the outlet temperature is 30°C, the feeding rate is 50 kg / h, and the pressure of the atomizer is 15 MPa. Obtain the dried powder of hemp seed protein peptides and use a sterile aluminum foil bag for vacuum-sealed packaging.

[0031] Sterilize the finished product of hemp seed protein peptides using an ultra-high pressure sterilization autoclave. The working pressure is 100 - 500 MPa, the working temperature is 25 - 40°C, the pressure holding time is 20 - 90 s, and store at room temperature after sterilization.

[0032] Test Examples 1 - 3 to obtain the yield of hemp seed protein peptides, α-amylase inhibitory activity, and α-glucosidase inhibitory activity. The data is shown in Table 1.

[0033] Table 1 Data of the yield of hemp seed protein peptides, α-amylase inhibitory activity, and α-glucosidase inhibitory activity in Examples 1 - 3

[0034] As can be seen from Table 1, the hemp seed protein peptides prepared in Examples 1 - 3 all have the effect of blocking sugar absorption. Among them, the hemp seed protein peptides prepared with the specific parameter values in Example 3 have the highest yield, and the best inhibitory activities against α-amylase and glucosidase, preliminarily proving that the process for preparing hemp seed protein peptides with sugar absorption blocking activity by this method is the optimal technological process.

[0035] Test the yield of hemp seed protein peptides, oil content, and sample storage stability of the hemp seed protein peptides prepared in Example 3 and Comparative Examples 1 - 2. The data is shown in Table 2.

[0036] Table 2 Data of the yield of hemp seed protein peptides, oil content, and sample storage stability detection of Example 3 and Comparative Examples 1 - 2

[0037] As can be seen from Table 2, the low-temperature continuous phase change degreasing, and the stepwise hydrolysis with polysaccharide enzyme and protease used in the present invention have certain effects on the yield, oil content, and sugar content of hemp seed protein peptides.

[0038] Perform mass spectrometry analysis, in vitro enzyme activity inhibition experiments, and clinical experiments on the hemp seed protein peptides obtained based on Example 3 to verify their sugar absorption blocking activity: 1. Perform mass spectrometry analysis on the dried powder of hemp seed protein peptides using HPLC-MS. The specific parameters and steps are as follows: Use a reverse-phase chromatographic column, with an aqueous phase of 0.1% formic acid aqueous solution and an organic phase of acetonitrile as the mobile phase, a flow rate of 0.2 - 1.0 ml / min, a column temperature of 25 - 40 °C, an ESI spray voltage of 2.5 - 5 kV, nitrogen as the gas, a gas pressure of 1 - 3 Pa, and a collision energy of 10 - 50 eV. The core peptide segments are as follows: SEQ ID NO: 1: Met-Thr-Met-Trp, SEQ ID NO: 2: Tyr-Lys-Pro-Val-Tyr, SEQ ID NO: 3: Thr-Pro-Val-Ser-Ile-Leu, SEQ ID NO: 4: Met-Ala-His-Leu-Phe, SEQ ID NO: 5: Met-Val-Ser-Leu-Tyr.

[0039] 1. α-Amylase and glucosidase inhibitory activities 1.1 Determination of α-amylase inhibitory activity Figure 1 This is a graph for the determination of α-amylase inhibitory activity. The IC50 of hemp seed protein peptides measured in the experiment is 4.975 mg / mL. It can be Figure 1 observed that the inhibition rate of hemp seed protein peptides on α-amylase activity reaches the highest (84.2%) at a concentration of 4 mg / mL. The results show that hemp seed protein peptides have certain inhibitory activity on amylase, which can hinder the hydrolysis and digestion of carbohydrates in food, thereby reducing the body's intake of sugar.

[0040] 1.2 Determination of glucosidase inhibitory activity Figure 2 This is a graph for the determination of α-glucosidase inhibitory activity. The IC50 of hemp seed protein peptides measured in the experiment is 0.53 mg / mL, while the IC50 of hemp seed protein is too large to be calculated by software. It can be Figure 2 observed that the inhibition rate of hemp seed protein peptides on α-glucosidase activity reaches the highest (75.1%) at a concentration of 4 mg / mL, which is much higher than the inhibition rate of hemp protein on α-glucosidase, indicating that hemp seed protein peptides at an appropriate concentration can effectively delay the absorption of carbohydrates and reduce postprandial hyperglycemia.

[0041] 2. Clinical trials Volunteer recruitment The inclusion criteria were as follows: healthy male or female volunteers; age, 18 to 55 years old; body mass index, 18 to 25 kg / m2 (healthy group) or BMI > 25 (overweight group); no previous diagnosis of diabetes or prediabetes; fasting blood glucose, 3.9 to 5.5 mmol / L (70 - 100 mg / dL) (healthy group) or fasting blood glucose greater than 6.0 (hyperglycemic group); not taking any medications for the treatment of diabetes or insulin resistance; and willing to provide written informed consent to participate in the study.

[0042] The exclusion criteria were as follows: abnormal results in liver function tests (alanine aminotransferase and aspartate aminotransferase), kidney function tests (blood urea nitrogen and creatinine), complete blood count, urine analysis, fecal occult blood test, or electrocardiogram; pregnant or lactating women; recent participation in clinical trials (within the last 3 months); history of malignant tumors or current malignant tumors; history of drug dependence, substance abuse, or alcoholism; history of drug or food allergies or allergies to any components of the test product; history of primary diseases of known major organs (such as the heart, liver, or kidneys), or history of digestive, metabolic, neurological, or mental diseases; taking medications known to cause organ damage within the previous 3 months; and taking any medications within the past 2 weeks.

[0043] 2.2, Test operation procedures All volunteers were advised to avoid alcohol or strenuous physical activity the day before. A SanNuo continuous blood glucose monitor was installed before the trial to ensure that all volunteers' instruments were activated and the data was stable, and a family group was established. The formal experiment began the next day. Volunteers fasted for at least 10 hours (allowed to drink water) before the trial, and the testing started at around 8:00 am on the second to fifth days. Volunteers ate a bowl of spinach and egg white porridge, 1 slice of white bread, and a small amount of pickled mustard within 10 minutes. At the same time, 2 g, 5 g, 10 g, and 1 g of hemp seed protein peptide were taken orally in sequence every day.

[0044] The second round of testing started after the post - lunch blood glucose returned to the baseline every afternoon. Each person drank a large cup of Starbucks Golden Baked Peach Oat Latte every day, and at the same time, 2 g, 5 g, 10 g, and 1 g of hemp seed protein peptide were ingested in sequence every day.

[0045] After each test, the participants fasted for 2 hours without eating food or drinking water. Blood glucose at 150 minutes after meals of the volunteers was collected through the "Love Health" app family group, and data was collected every 3 minutes.

[0046] 2.3, Area under the curve of post - meal blood glucose response and statistical analysis The blood glucose curve at 120 minutes after meals was plotted, and the area under the curve (AUC) was calculated using Graphpad (9.0) software. Repeated - measures one - way analysis of variance and paired T - tests were used to compare the AUC, blood glucose peak value, time to peak, and maximum blood glucose increase between each intervention and the baseline ( Figure 3Schematic diagram of postprandial blood glucose fluctuation parameters for continuous blood glucose monitoring). The differences between different dose treatments and the baseline were analyzed by paired-sample T test.

[0047] Figure 4 For continuous blood glucose monitoring of post-breakfast blood glucose fluctuation (8 people in each group, 2 groups in total). By Figure 4 It can be seen that the test subjects in the experimental group consumed hemp seed protein peptide while consuming white porridge and bread, and had a smaller degree of blood glucose fluctuation compared with the control group test subjects who only consumed white porridge and bread. The statistical analysis results of all subjects showed that: consuming 1 g of hemp seed protein peptide after consuming white porridge and bread had a certain effect on blood glucose peak value and peak area (an average decrease of 30%); after consuming 2 g of hemp seed protein peptide, the blood glucose peak values of 5 test subjects decreased significantly (an average decrease of 38%), and the blood glucose peak area decreased significantly (an average decrease of 49%); after consuming 5 g of hemp seed protein peptide, the blood glucose peak values of 6 out of 8 test subjects decreased significantly (an average decrease of 67%), and the blood glucose peak area of these 5 test subjects decreased significantly (an average decrease of 56%).

[0048] By Figure 5 For continuous blood glucose monitoring of post-afternoon tea blood glucose fluctuation (8 people in each group, 2 groups in total). It can be seen that the test subjects in the experimental group consumed hemp seed protein peptide while consuming milk tea, and had a smaller degree of blood glucose fluctuation compared with the control group test subjects who only consumed milk tea. The statistical analysis results of all subjects showed that: after consuming milk tea, when consuming 2 g and 5 g of hemp seed protein peptide, the blood glucose fluctuation within 150 min after the meal decreased, the blood glucose peak value decreased (the average decreases were 41.8% and 44.9% respectively), and the area under the blood glucose curve decreased (the average decreases were 61% and 58% respectively).

[0049] In summary, the in vitro enzyme activity inhibition test and the human clinical trial results show that: the hemp seed protein peptide obtained by the technical solution and preparation method of the present invention can significantly inhibit the activities of α-amylase and α-glucosidase, thereby blocking the digestion and absorption of starch and sucrose by the human body. The clinical test results show that the hemp seed protein peptide prepared by the present invention can effectively reduce the postprandial blood glucose peak value and peak area, and the effective sugar-breaking rate can reach 30 - 60%.

[0050] The above are the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art in this technical field, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of hemp seed protein peptide with the function of blocking sugar absorption, characterized in that, It includes the following steps: Crush the hemp seeds, degrease them through low-temperature continuous phase transition to obtain defatted hemp meal. The defatted hemp meal is successively hydrolyzed by polysaccharide enzyme and protease. After the hydrolysis is completed, low-temperature salting out is carried out, followed by drying and sterilization to obtain hemp seed protein peptides.

2. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The extractant for the low-temperature continuous phase transition is solvent oil No. 15; the mass-volume ratio of the hemp seeds to the extractant is 1:8 - 15; the low-temperature continuous phase transition includes the following conditions: the temperature is 20°C - 35°C, the pressure is 2.5 Mpa - 4 Mpa, the flow rate is 50 - 80 L / h, the continuous extraction time is 90 min - 120 min, the desorption temperature is 70°C - 85°C, and the desorption pressure is 0.5 Mpa - 1 Mpa.

3. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The polysaccharide enzyme hydrolysis includes the following steps: Mix the defatted hemp meal with water, with a material-liquid ratio of 1:10 - 30. The mass of the added polysaccharide enzyme is 10 - 15‰ of the substrate mass. Adjust the pH to 3.0 - 5.5, and the hydrolysis duration is 1 - 4 h; the substrate is the defatted hemp meal and water.

4. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The polysaccharide enzyme is one or a combination of two or more of cellulase, pectinase, amylase, glycosidase, hemicellulose, glucanase, and xylanase.

5. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The protease hydrolysis includes the following steps: Inactivate the enzyme in the product after polysaccharide enzyme hydrolysis, then add protease. The addition amount of the protease is 2 - 6‰ of the mass of the product after polysaccharide enzyme hydrolysis. Adjust the pH to 6.0 - 8.5 for hydrolysis, and the hydrolysis duration is 15 - 35 min; the protease is bacillus licheniformis protease.

6. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The low-temperature salting out is to centrifuge and filter the product after protease hydrolysis to obtain a filtrate. Cool it to 2 - 4°C, continuously add saturated ammonium sulfate solution to the filtrate until protein flocculation occurs, carry out salting out for 30 - 90 min, and take the precipitate after centrifugation.

7. The preparation method of the hemp seed protein peptide with a sugar absorption blocking effect according to claim 1, characterized in that, The drying is spray drying, including the following steps: Use a spray dryer for spray drying. The inlet air temperature is 15 - 25°C, the outlet air temperature is 30 - 40°C, the feeding rate is 50 - 150 kg / h, the voltage of the electrostatic generator is 30 - 55 kV, and the pressure of the atomizer is 15 - 20 MPa.

8. The preparation method of the hemp seed protein peptide with the function of blocking sugar absorption according to claim 1, characterized in that, The sterilization is ultra-high pressure sterilization, including the following steps: During ultra-high pressure sterilization, the working pressure is 100 - 500 MPa, the working temperature is 25 - 40°C, and the pressure holding time is 20 - 90 s.

9. A hemp seed protein peptide prepared by the preparation method of the hemp seed protein peptide with a sugar absorption blocking effect according to any one of claims 1 to 8, characterized in that, It includes the following short peptide sequences: SEQ ID NO: 1: Met-Thr-Met-Trp, SEQ ID NO: 2: Tyr-Lys-Pro-Val-Tyr, SEQ ID NO: 3: Thr-Pro-Val-Ser-Ile-Leu, SEQ ID NO: 4: Met-Ala-His-Leu-Phe, SEQ ID NO: 5: Met-Val-Ser-Leu-Tyr.

10. Use of the hemp seed protein peptides prepared by the preparation method of the hemp seed protein peptides with a sugar absorption blocking effect as described in claim 9 in sugar absorption blocking foods.

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